Membrane proteins are divided into two groups based upon the ease with which the proteins can be removed from the membrane. Extrinsic or peripheral membrane proteins can be removed using extremes of ionic strength or pH, or urea or other disruptors of protein interactions. Intrinsic or integral membrane proteins are released only when the lipid bilayer of the membrane is dissolved by detergent.
The majority of known integral membrane proteins are transmembrane proteins which are characterized by an extracellular, a transmembrane, and an intracellular domain. Transmembrane proteins are typically embedded into the cell membrane by one or more regions comprising 15 to 25 hydrophobic amino acids which are predicted to adopt an .alpha.-helical conformation. Transmembrane proteins are classified as bitopic (or Types I and II) and polytopic (or Types III and IV) [Singer, S. J. (1990) Annu. Rev. Cell Biol. 6:247-96]. Bitopic proteins span the membrane once while polytopic proteins contain multiple membrane-spanning segments. Type III integral membrane proteins have multiple transmembrane stretches of hydrophobic residues. Transmembrane proteins carry out a variety of important cellular functions such as acting as cell-surface receptor proteins which are involved in signal transduction (e.g., growth factor receptors) and transport of ions or metabolites (e.g., ion channels).
Recently a multigene family encoding type III integral membrane proteins, termed the transmembrane 4 superfamily (TM4SF) or Tetraspan family, was identified [Wright, M. D. and Tomlinson, M. G. (1994) Immunol. Today 15:588]. The TM4SF comprises a superfamily of membrane proteins which cross or traverse the cell membrane four times. Members of the TM4SF include a number of platelet and endothelial cell membrane proteins [PETA-3, CD9 (lung adenocarcinoma antigen MRP-1), the platelet and melanoma-associated antigen CD63 (ME491)], leukocyte surface glycoproteins [CD53, CD37, CD63, R2, and the tumor associated antigen TAPA-1 (CD81)], the colonal carcinoma antigen CO-029, mink lung epithelial protein TI-1, the tumor-associated antigen L6, SAS (a gene amplified in human sarcomas), the product of a gene responsible for slow retinal degeneration in mice (the rds gene product), and surface proteins of the schistosome parasites [Fitter, S. et al. (1995) Blood 86:1348; Bouchiex et al. (1991) J. Biol. Chem. 266:117; Wright, M. D. et al. (1993), Int. Immunol. 5:209; Classon, B. J. (1989) J. Exp. Med. 169:1497 and (1990) J. Exp. Med. 172:1007; Hotta, J. et al. (1988) Cancer Res. 48:2955; Gaugitsch, T. et al. (1991) Eur. J. Immunol. 21:377; Oren, R. et al. (1990) Mol. Cell. Biol. 10:4007; Szala, S. et al. Proc. Natl. Acad. Sci. USA 87:6833; Kallin, B. et al. (1991) Mol. Cell. Biol. 11:5338; Miyake, M. (1991) J. Exp. Med. 174:1347; Marken, J. S. et al. (1992) Proc. Natl. Acad. Sci. USA 89:3503; Travis, G.H. et al. (1991) Nature 338:70; Jankowski, S. A. (1994) Oncogene 9:1205; and Wright, M. D. et al. (1990) J. Immunol. 144:3195]. The members of the TM4SF show about 25-30% amino acid sequence identity with one another.
The predicted structure of the TM4SF proteins reveals a topology where the N- and C-termini are intracellular and the major hydrophilic domain, located between transmembrane domains 3 and 4, is extracellular. TM4SF members are most conserved in their transmembrane and cytoplasmic domains (the conservation among transmembrane domains being the highest) and most divergent in their two hydrophilic extracellular domains. The high level of conservation seen in the transmembrane and cytoplasmic domains of TM4SF members suggest an effector/signaling function common to all members. The divergence of the extracellular domains suggests these domains provide functions specific to each family member such as ligand binding or protein--protein interaction [Wright, M. D. et al. (1993), supra and Fitter, S. et al., supra].
A number of TM4SF members have been implicated in signal transduction, control of cell adhesion, and regulation of cell growth and proliferation (including development and oncogenesis) and motility (including the ability to suppress metastatic potential) and expression of a number of TM4SF members is associated with a variety of tumors (e.g., CD81/TAPA-1, L6, CD9/MRP-1, L6, CD63/ME491, CO-029, SAS, PETA-3). The expression of several TM4SF members is altered when cells are growing or activated. CD9, CD53, and CD82 are upregulated when lymphocytes are activated. Cell surface expression of CD37 is rapidly lost upon activation of B cells. Other TM4SF members are implicated in cell growth due to their association with tumor cells. CD9 (MRP-1) is a marker for 90% of non-T acute lymphoblastic leukemia cells and 50% of acute myeloid and chronic lymphoid leukemias; CD9 is not expressed on resting B and T lymphocytes. Anti-CD9 antibodies inhibit the motility of a variety of cancer cell lines and inhibit the metastatic potential of the mouse BL6 cell line, a highly metastatic variant of B16 cells [Miyake, M. and Hakomori, S. (1991) Biochem. 30:3328]. Expression of CD9 in transfection experiments correlated with suppression of metastatic potential and cell motility [Ikeyama, S. et al. (1993) J. Exp. Med. 174:1347]. CD63 (ME491) is expressed in early stage melanoma but is downregulated in advanced stages of melanoma; CD63 is not expressed on normal tissue melanocytes. CO-029 is expressed on colon, gastric, pancreatic, and rectal carcinomas but not on most normal tissues. The gene encoding SAS is amplified in a subset of human sarcomas.
The tumor associated antigen L6 has been classified as a distant member of the TM4SF multigene family [Marken et al. (1992) Proc. Natl. Acad. Sci. USA 89:3503]. The L6 antigen is a hydrophobic, cysteine-rich integral membrane protein that shares a similar structure with members of the TM4SF family (e.g., three closely spaced N-terminal hydrophobic or transmembrane domains, a hydrophilic extracellular domain and a C-termninal hydrophobic domain). Homologs of the L6 antigen have been identified in mice, hamsters, and humans [Marken et al. (1994) J. Biol. Chem. 269:7397; Marken et al. (1992), supra]. L6 is highly expressed on a number of human carcinomas, including lung, breast, colon, and ovarian carcinomas [Hellstrom et al. (1986) Cancer Res. 46:3917]. L6 is also expressed at low levels on some normal human tissues, endothelial cells in particular [DeNardo et al. (1991) Nucl. Med. Biol. 18:621]. The very high expression of L6 on tumors has led to clinical studies employing anti-L6 antibodies. A murine monoclonal anti-L6 antibody (mAb L6) administered in a phase I study to patients with recurrent cancers of the breast, lung, colon or ovary demonstrated that the mAb L6 effectively localized to the tumor, was well tolerated, and, in one patient, induced a complete, although temporary, remission [Goodman et al. (1990) J. Clin. Oncol. 8:1083]. In other clinical studies, anti-L6 antibodies were labeled with .sup.125 I and were shown to deliver therapeutic amounts of radioactivity to tumors [DeNardo et al. (1991) In Breast Epithelial Antigens, Ceriani (ed.), Plenum, N.Y., pp. 227-232]. L6 has been postulated to play a role in cell growth and the over expression and/or ectopic expression of L6 may result in tumor growth as is the case for other TM4SF family members (e.g., CD63/ME491, CO-029).
The discovery of molecules related to the TM4SF multigene family to general, and in the L6 family in particular, satisfies a need in the art by providing new diagnostic or therapeutic compositions useful in the treatment of disorders associated with alterations in the expression of members of the TM4SF multigene family.